Using a Xenopus expression-cloning screen, we have isolated Gremlin, a novel antagonist of bone morphogenetic protein (BMP) signaling that is expressed in the neural crest. Gremlin belongs to a novel gene family that includes the head-inducing factor Cerberus and the tumor suppressor DAN. We show that all family members are secreted proteins and that they act as BMP antagonists in embryonic explants. We also provide support for the model that Gremlin, Cerberus, and DAN block BMP signaling by binding BMPs, preventing them from interacting with their receptors. In addition, Cerberus alone blocks signaling by Activin- and Nodal-like members of the TGF beta superfamily. Therefore, we propose that Gremlin, Cerberus, and DAN control diverse processes in growth and development by selectively antagonizing the activities of different subsets of the TGF beta ligands.

During limb outgrowth, signaling by bone morphogenetic proteins (BMPs) must be moderated to maintain the signaling loop between the zone of polarizing activity (ZPA) and the apical ectodermal ridge (AER). Gremlin, an extracellular Bmp antagonist, has been proposed to fulfill this function and therefore be important in limb patterning. We tested this model directly by mutating the mouse gene encoding gremlin (Cktsf1b1, herein called gremlin). In the mutant limb, the feedback loop between the ZPA and the AER is interrupted, resulting in abnormal skeletal pattern. We also show that the gremlin mutation is allelic to the limb deformity mutation (ld). Although Bmps and their antagonists have multiple roles in limb development, these experiments show that gremlin is the principal BMP antagonist required for early limb outgrowth and patterning.

Zero-mode waveguides provide a powerful technology for studying single-molecule real-time dynamics of biological systems at physiological ligand concentrations. We customized a commercial zero-mode waveguide-based DNA sequencer for use as a versatile instrument for single-molecule fluorescence detection and showed that the system provides long fluorophore lifetimes with good signal to noise and low spectral cross-talk. We then used a ribosomal translation assay to show real-time fluidic delivery during data acquisition, showing it is possible to follow the conformation and composition of thousands of single biomolecules simultaneously through four spectral channels. This instrument allows high-throughput multiplexed dynamics of single-molecule biological processes over long timescales. The instrumentation presented here has broad applications to single-molecule studies of biological systems and is easily accessible to the biophysical community.D etermining the molecular details of the time evolution of complex multicomponent biological systems requires analysis at the single-molecule level because of their stochastic and heterogeneous nature. Ideally, such experiments would track simultaneously the composition of a biological system (bound ligands, factors, and cofactors) and the conformation of the individual molecules in real time. Single-molecule fluorescence methods, such as total internal reflection fluorescence (TIRF) microscopy, allow the observations of the compositional dynamics (through arrival of fluorescently labeled ligands, factors, or cofactors) and conformational dynamics (through FRET) of single-molecular species. However, these traditional singlemolecule methods are hindered by limitations in maximal fluorescent component concentrations (up to 50 nM) (1), limited simultaneous detection (two to three colors) (2-6), and low throughput (a few hundred molecules at most per experiment) (7). As such, the full potential of single-molecule fluorescence to investigate a range of biological problems under physiologically relevant conditions has not yet been harnessed.Zero-mode waveguides (ZMWs) are small metallic apertures patterned on glass substrates that overcome the concentration restrictions by optically limiting background excitation (8). Each ZMW consists of an ∼150-nm-diameter metallic aperture that restricts the excitation light to a zeptoliter volume, making possible experiments with near-physiological concentrations (up to 20 μM) of fluorescently labeled ligands (1). Previous advances in nanofabrication (9), surface chemistry (10), and detection instrumentation (11) have led to ZMW-based instrumentation capable of the direct observation of DNA polymerization (12), reverse transcription (13), processive myosin motion (14), and translation by the ribosome (15, 16) with multicolor single-molecule detection. However, this sophisticated technology has not been broadly available to the scientific community. Despite multiple efforts to develop ZMW instrumentation, the combined difficulties in fabrica...

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The need to regulate X chromosome expression in Caenorhabditis elegans arises as a consequence of the primary sex-determining signal, the X/A ratio (the ratio of X chromosomes to sets of autosomes), which directs 1X@A animals to develop as males and 2X/2A animals to develop as hermaphrodites. C. elegans possesses a dosage compensation mechanism that equalizes X chromosome expression between the two sexes despite their disparity in X chromosome dosage. Previous genetic analysis led to the identification of four autosomal genes, dpy-21, dpy-26, dpy-27 and dpy-28, whose products are essential in XX animals for proper dosage compensation, but not for sex determination. We report the identification and characterization of dpy-30, an essential component of the dosage compensation machinery. Putative null mutations in dpy-30 disrupt dosage compensation and cause a severe maternal-effect, XX-specific lethality. Rare survivors of the dpy-30 lethality are dumpy and express their X-linked genes at higher than wild-type levels. These dpy-30 mutant phenotypes superficially resemble those caused by mutations in dpy-26, dpy-27 and dpy-28; however, detailed phenotypic analysis reveals important differences that distinguish dpy-30 from these genes. In contrast to the XX-specific lethality caused by mutations in the other dpy genes, the XX-specific lethality caused by dpy-30 mutations is completely penetrant and temperature sensitive. In addition, unlike the other genes, dpy-30 is required for the normal development of XO animals. Although dpy-30 mutations do not significantly affect the viability of XO animals, they do cause them to be developmentally delayed and to possess numerous morphological and behavioral abnormalities. Finally, dpy-30 mutations can dramatically influence the choice of sexual fate in animals with an ambiguous sexual identity, despite having no apparent effect on the sexual phenotype of otherwise wild-type animals. Paradoxically, depending on the genetic background, dpy-30 mutations cause either masculinization or feminization, thus revealing the complex regulatory relationship between the sex determination and dosage compensation processes. The novel phenotypes caused by dpy-30 mutations suggest that in addition to acting in the dosage compensation process, dpy-30 may play a more general role in the development of both XX and XO animals.